NMR - method suited to study biomolecules Pavel Kadeřávek 1. NMR - introduction 2. Studies of interactions 3. Conformational exchange 4. Investigation ps-ns dynamics of structured proteins 5. High-resolution relaxometry - investigation of ps-ns dynamics of IDPs 6. High-resolution relaxometry - metabolomics 7. Two-field NMR 8. Dissolution dynamics nuclear polarisation (dDNP) 1. NMR - introduction 2. Studies of interactions 3. Conformational exchange 4. Investigation ps-ns dynamics of structured proteins 5. High-resolution relaxometry - investigation of ps-ns dynamics of IDPs 6. High-resolution relaxometry - metabolomics 7. Two-field NMR 8. Dissolution dynamics nuclear polarisation (dDNP) NMR introduction • magnetic moments of nuclei (spin) - 1H, 1^C, lbN, J1P • Larmor frequencies uo = —jB (we need strong B) • slow relaxation polarisation transfer Effect of electron density -CH=0 -C=CH--CH2-0--CH2-C=0 -CH2-C- (CH3)4Si H2 _I I 0 ppm 10 ppm 20 ppm 30 ppm 1950: Proctor, Yu 1H NMR spectrum of a protein 1956 - first NMR spectrum of a protein Structure information Nuclear Overhauser effect: Spin-spin scalar coupling: distance torsion angles j 1985: first protein structure solved by NMR Residual dipolar couplings - Structure information 1995: Prestegard J.H., 1996 Bax A. 1. NMR 2. Studies of interactions 3. Conformational exchange 4. Investigation ps-ns dynamics of structured proteins 5. High-resolution relaxometry - investigation of ps-ns dynamics of IDPs 6. High-resolution relaxometry - metabolomics 7. Two-field NMR 8. Dissolution dynamics nuclear polarisation (dDNP) Interaction: titration figure copied from Charlier C. et al., J. Am. Chem. Soc, 2017, 139, 1219-12227 Interaction: titration figures copied from publications: Dicks et al., BMC Moleculart and Cell Biology, 2019, 20: 23 Waudby at al., Scientific reports, 2016, 24826 Interaction of small molecule with a large molecule selective saturation Wf'm |A h H h II II II I it li 11 1 , lAa off-resonance on-resonance difference spectra copied from publication: Addino Viegas et al., J. Chem. Educ, 2011, 88, 990-994 1. NMR 2. Studies of interactions 3. Conformational exchange 4. Investigation ps-ns dynamics of structured proteins 5. High-resolution relaxometry - investigation of ps-ns dynamics of IDPs 6. High-resolution relaxometry - metabolomics 7. Two-field NMR 8. Dissolution dynamics nuclear polarisation (dDNP) Conformational Exchange = Structure Rearrangement ®c g e g gggg@ g © qö pa- pb = 1 - pa /ys—ms motions kex < Qa - Q a - Rex(kex, ^a> ^b> Pb) A B k ex G © ßö pa- pb = 1 - pa /ys—ms motions kex > Qa - Q a - Rex(kex, ^a> ^b> Pb) A B k ex G © ßG ^a ^obs b pa> pb = 1 - pa Conformational Exchange = Structure Rearrangement Chemical Exchange Saturation Transfer Chemical Exchange Saturation Transfer Chemical Exchange Saturation Transfer Chemical Exchange Saturation Transfer Chemical Exchange Saturation Transfer Chemical Exchange Saturation Transfer Chemical Exchange Saturation Transfer Chemical Exchange Saturation Transfer Chemical Exchange Saturation Transfer Chemical Exchange Saturation Transfer Residual Dipolar Coupling Vallurupalli P. et al., Proc. Natl. Acad. Sei., 2007, 104, 18473-18477 1. NMR 2. Studies of interactions 3. Conformational exchange 4. Investigation ps-ns dynamics of structured proteins 5. High-resolution relaxometry - investigation of ps-ns dynamics of IDPs 6. High-resolution relaxometry - metabolomics 7. Two-field NMR 8. Dissolution dynamics nuclear polarisation (dDNP) RNA polymerase DNA • Gram positive bacteria Transcription 9 Bacillus subtilis RNA polymerase • RNA polymerase core: _.. _ ol ol 3 3 (jj RNA ^ ^ Translation Ribosom t PROTEIN S subunit important for virulency 2 domain protein: N-terminal domain: structured C-terminal domain: disordered MGIKQYSQEE LKEMALVEIA HELFEEHKKP VPFQELLNEI ASLLGVKKEE LGDRIAQFYT DLNIDGRFLA LSDQTWGLRS WYPYDQLDEE TQPTVKAKKK KAKKAVEEDL DLDEFEEIDE DDLDLDEVEE ELDLEADDFD EEDLDEDDDD LEIEEDIIDE DDEDYDDEEE EIK residue number 1. NMR 2. Studies of interactions 3. Conformational exchange 4. Investigation ps-ns dynamics of structured proteins 5. High-resolution relaxometry - investigation of ps-ns dynamics of IDPs 6. High-resolution relaxometry - metabolomics 7. Two-field NMR 8. Dissolution dynamics nuclear polarisation (dDNP) Intrinsically disordered protein (IDP) • intrinsically disordered protein (IDP) - lack of stable 3D structure - high flexibility - structural adaptability - polyfunctionality figure copied from: softsimu,blogspotir/2013/preformed-structural-elements-in-long.html Intrinsically disordered protein (IDP) - challenges Free energy landscape B c Rigid protein Multiple conformations IDP figure copied from: M. Bruscale, B. Schuler, B. Samori, Chem. Rev., 2014, vol. 114, 3281 Intrinsically disordered protein (IDP) - challenges • complex motion - distribution of timescales • NMR relaxation at different magnetic fields - sensitive to various frequencies • low frequencies =>■ low Bo = low resolution 10 r/ns High-resolution relaxometry Br Bore of the spectrometer Area of homogeneou high magnetic field Sample High-field probe High-resolution relaxometry Bore of the spectrometer Area of homogeneou high magnetic field Sample High-field probe High-resolution relaxometry Bore of the spectrometer Area of homogeneou high magnetic field Sample High-field probe High-resolution relaxometry Bore of the spectrometer Area of homogeneou high magnetic field Sample High-field probe High-resolution relaxometry - device NMR spectrometer Sample shuttle tube Triple resonance probe pneumatic shuttling: 0.5 m in ~120ms Charlier C. et al., J. Am. Chem. Soc, 2013, 135 (49), 18665-18672 delta subunit: longitudinal relaxation rate 10 14.10T C/3 DC 8 h 6 h 4 h 2 h •** * * *** ***** 0 80 90 100 110 120 130 residue number 140 150 160 170 delta subunit: relaxometry relaxation rate 14.10T i—i—i - 4.00 T i—i—i **** *** **** i *** * i i i i *** ***** i i i 80 90 100 110 120 130 140 150 160 170 residue number delta subunit: relaxometry relaxation rate 10 14.10T i—i—i 4.00 T i—i—i 1.00 T i—i—i 8 h 6 h I T " 111 %*% I* 4 h 2 h •** * *** »♦»»*♦»»»* ***** ** 0 80 90 100 110 120 130 140 150 residue number 160 170 delta subunit: distribution of motions 0.6 0.5 CM 0.4 < t- 0.3 0.2 0.1 0.0 < 10"1 ns 1 80 90 100 110 120 130 140 150 160 170 residue number CO < 0.6 0.5 0.4 0.3 0.2 0.1 0.0 - 10"1 ns ill il,i.lii,iIIII1M 80 90 100 110 120 130 140 150 160 170 residue number 0.6 0.5 0.4 < 0.3 0.2 0.1 0.0 -10° ns 11 ml 80 90 100 110 120 130 140 150 160 170 residue number 0.6 0.5 0.4 < 0.3 0.2 0.1 0.0 > 10° ns 1 As 80 90 100 110 120 130 140 150 160 170 residue number 81____DQLDEE TQPTVKAKKK KAKKAVEEDL DLDEFEEIDE DDLDLDEVEE ELDLEADDFD EEDLDEDDDD LEIEEDIIDE DDEDYDDEEE EIK 1. NMR 2. Studies of interactions 3. Conformational exchange 4. Investigation ps-ns dynamics of structured proteins 5. High-resolution relaxometry - investigation of ps-ns dynamics of IDPs 6. High-resolution relaxometry - metabolomics 7. Two-field NMR 8. Dissolution dynamics nuclear polarisation (dDNP) Metabolomics • investigation of metabolites in biological fluids • substrates and products of enzymatic reactions • cofactors or regulators copied from publication: Ziqing Wang et al., J. Am. Chem. Soc, 2021, 143, 9393-9404 Metabolomics • dependence of relaxation rate on rotational diffusion • Stokes law =^> size of the molecule free x bound 10 5 0.01 0.1 1 Magnetic field (T) 10 12 10 ■P 8 t£ 6 4 - Alanine TSP ■ _J—L 0.1 1 Magnetic field (T) copied from publication: Ziqing Wang et al., J. Am. Chem. Soc, 2021, 143, 9393-9404 Metabolomics 106 10s 10"4 Concentration (mol/L) copied from publication: Ziqing Wang et al., J. Am. Chem. Soc, 2021, 143, 9393-9404 Metabolomics competition between ligands 0.1 1 10 0.1 1 10 Magnetic field (T) Magnetic field (T) copied from publication: Ziqing Wang et al., J. Am. Chem. Soc, 2021, 143, 9393-9404 1. NMR - introduction 2. Studies of interactions 3. Conformational exchange 4. Investigation ps-ns dynamics of structured proteins 5. High-resolution relaxometry - investigation of ps-ns dynamics of IDPs 6. High-resolution relaxometry - metabolomics 7. Two-field NMR 8. Dissolution dynamics nuclear polarisation (dDNP) TOCSY TOCSY = total correlation spectroscopy: • information about bonds • requires high irradiation suppressing effects of chemical shift • stronger magnetic field = stronger irradiation C62 C61 CY Ca Ca C3 Cy C61C62 TOCSY 50 • E Q_ Q-U 100 no i—i i # H K) 150 High-field TOCSY • * 150 100 50 (5,-13C (ppm) Two-field NMR spectrometer NMR spectrometer Sample shuttle tube Triple resonance probe pneumatic shuttling: 0.5 m in ~120ms Cousin S.F. et al., Phys Chem Chem Phys, 2016, 18 (48), 33187-33194 TOCSY at two-fields š j tí • ♦> • * 50 » * o 4» 6 o set 0 • (ppm) u 100 m i—i 1 • t i 1 * 150 2F • • -TOCSY O Q • a o t>* T-1-1-1-1-1-1-I-1-1-1-1-1 I-1 I 150 100 50 Ô7-13C (ppm) TOCSY at two-fields 180 150 140 Ô2-13C (ppm) /ys—ms motions kex > Qa - Q a - Rex(kex, ^a> ^b> Pb) A B k ex G © ßG ^a ^obs b pa> pb = 1 - pa Exchange effects suppression at two-fields 1. NMR - introduction 2. Studies of interactions 3. Conformational exchange 4. Investigation ps-ns dynamics of structured proteins 5. High-resolution relaxometry - investigation of ps-ns dynamics of IDPs 6. High-resolution relaxometry - metabolomics 7. Two-field NMR 8. Dissolution dynamics nuclear polarisation (dDNP) Dissolution dynamics nuclear polarisation (dDNP) Temperature (K) copied from publication: Bertrand Plainchont et al., Anal. Chem., 2018, 90, 3639-3650 Dissolution dynamics nuclear polarisation (dDNP) r Magnetic Tunnel copied from publication: Bertrand Plainchont et al., Anal. Chem., 2018, 90, 3639-3650 Dissolution dynamics nuclear polarisation (dDNP) copied from publication: Sami Jannin et al., J. Mag. Reson., 2019, 305, 41-50